Vaccines have been utilized to provide a long term protection against a number of disease conditions by very limited administration of a prophylactic agent that stimulates an organism's immune system to destroy disease pathogens before they can proliferate and cause a pathological effect. Various approaches to vaccines and vaccinations are described in Bernard R. Glick and Jack J. Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA, Second Edition, ASM Press pp. 253-276 (1998).
Vaccination is a means of inducing the body's own immune system to seek out and destroy an infecting agent before it causes a pathological response. Typically, vaccines are either live, but attenuated, infectious agents (virus or bacteria), or a killed form of the agent. A vaccine consisting of a live bacteria or virus must be non-pathogenic. Typically, a bacterial or viral culture is attenuated (weakened) by physical or chemical treatment. Although the agent is nonvirulent, it can still elicit an immune response in a subject treated with the vaccine.
An immune response is elicited by antigens, which can be either specific macromolecules or an infectious agent. These antigens are generally either proteins, polysaccharides, lipids, or glycolipids, which are recognized as “foreign” by lymphocytes known as B cells and T cells. Exposure of both types of lymphocytes to an antigen elicits a rapid cell division and differentiation response, resulting in the formation of clones of the exposed lymphocytes. B cells produce plasma cells, which in turn, produce proteins called antibodies (Ab), which selectively bind to the antigens present on the infectious agent, thus neutralizing or inactivating the pathogen (humoral immunity). In some cases, B cell response requires the assistance of CD4 helper T cells.
The specialized T cell clone that forms in response to the antigen exposure is a cytotoxic T lymphocyte (CTL), which is capable of binding to and eliminating pathogens and tissues that present the antigen (cell-mediated or cellular immunity). In some cases, an antigen presenting cell (APC) such as a dendritic cell, will envelop a pathogen or other foreign cell by endocytosis. The APC then processes the antigens from the cells and presents these antigens in the form of a histocompatibility molecule:peptide complex to the T cell receptor (TCR) on CTLs, thus stimulating an immune response.
Humoral immunity characterized by the formation of specific antibodies is generally most effective against acute bacterial infections and repeat infections from viruses, whereas cell-mediated immunity is most effective against viral infection, chronic intracellular bacterial infection, and fungal infection. Cellular immunity is also known to protect against cancers and is responsible for rejection of organ transplants.
Antibodies to antigens from prior infections remain detectable in the blood for very long periods of time, thus affording a means of determining prior exposure to a pathogen. Upon re-exposure to the same pathogen, the immune system effectively prevents reinfection by eliminating the pathogenic agent before it can proliferate and produce a pathogenic response.
The same immune response that would be elicited by a pathogen can also sometimes be produced by a non-pathogenic agent that presents the same antigen as the pathogen. In this manner, the subject can be protected against subsequent exposure to the pathogen without having previously fought off an infection.
Not all infectious agents can be readily cultured and inactivated, as is required for vaccine formation, however. Modern recombinant DNA techniques have allowed the engineering of new vaccines to seek to overcome this limitation. Infectious agents can be created that lack the pathogenic genes, thus allowing a live, nonvirulent form of the organism to be used as a vaccine. It is also possible to engineer a relatively nonpathogenic organism such as E. coli to present the cell surface antigens of a pathogenic carrier. The immune system of a subject vaccinated with such a transformed carrier is “tricked” into forming antibodies to the pathogen. The antigenic proteins of a pathogenic agent can be engineered and expressed in a nonpathogenic species and the antigenic proteins can be isolated and purified to produce a “subunit vaccine.” Subunit vaccines have the advantage of being stable, safe, and chemically well defined; however, their production can be cost prohibitive.
A new approach to vaccines has emerged in recent years, broadly termed genetic immunization. In this approach, a gene encoding an antigen of a pathogenic agent is operably inserted into cells in the subject to be immunized. The treated cells, preferably antigen presenting cells (APCs) such as the dendritic cells, are transformed and produce the antigenic proteins of the pathogen. These in vivo-produced antigens then trigger the desired immune response in the host. The genetic material utilized in such genetic vaccines can be either a DNA or RNA construct. Often the polynucleotide encoding the antigen is introduced in combination with other promoter polynucleotide sequences to enhance insertion, replication, or expression of the gene.
DNA vaccines encoding antigen genes can be introduced into the host cells of the subject by a variety of delivery systems. These delivery systems include prokaryotic and viral delivery systems. For example, one approach is to utilize a viral vector, such as vaccinia virus incorporating the new genetic material, to innoculate the host cells. Alternatively, the genetic material can be incorporated in a plasmid vector or can be delivered directly to the host cells as a “naked” polynucleotide, i.e. simply as purified DNA. In addition, the DNA can be stably transfected into attenuated bacteria such as Salmonella typhimurium. When a patient is orally vaccinated with the transformed Salmonella, the bacteria are transported to Peyer's patches in the gut (i.e., secondary lymphoid tissues), which then stimulate an immune response.
DNA vaccines provide an opportunity to immunize against disease states that are not caused by traditional pathogens, such as genetic diseases and cancer. Typically, a genetic cancer vaccine introduces into APCs a gene that encodes an antigen, and the so transformed APCs produce antigens to a specific type of tumor cell. An effective general vaccine against a number of cancer types can thus entail numerous individual vaccines for each type of cancer cell to be immunized against.
Inhibitor of Apoptosis Proteins (i.e., IAP-family proteins) are a class of natural antigens expressed in many different tumor cells. As the name suggests, these proteins, in their natural form, inhibit apoptosis (i.e., programmed cell death), which in turn, may lead to resistance of cancer cells to apoptosis inducing chemotherapeutic agents, such as etoposide. Examples of IAP-family proteins include X chromosome-associated IAP (XIAP), NAIP, cIAP1 (also known as BIRC2), cIAP2 (also known as BIRC3), bruce (also known as BIRC6), survivin (also known as BIRC5), and livin (also known as BIRC7, KIAP, and ML-IAP). The mammalian IAP family of proteins includes proteins with three BIR domains (e.g., XIAP, cIAP1, cIAP2, and NAIP), as well as proteins with a single BIR domain (e.g., survivin and livin).
Tamm et al. Cancer Res. 1998; 58(23):5315-20, have reported expression of the human survivin in 60 human tumor cell lines. Tamm et al. have also reported that survivin and XIAP were both effective at inhibiting programmed cell death (apoptosis) induced by treatment of tumor cells with apoptosis inducing agents such as Bax or Fas (CD95). Survivin and other IAP-family proteins reportedly inhibit apoptosis by binding to effector cell death proteases, e.g., caspase-3 and caspase-7. Mutations in IAP-family proteins can lead to reduced apoptosis inhibition activity or even to apoptosis inducing activity relative to the activity of the wild-type IAP-family protein. The anti-apoptotic activity of the IAP-family proteins is believed to be associated with the BIR domain.
Survivin reportedly is present in most common human cancer cells, including cancers of the lung, prostate, breast, and pancreas. Survivin has also been identified in high-grade, non-Hodgkin's lymphomas, but not in low-grade non-Hodgkin's lymphomas. Reportedly, survivin is present in normal cells during fetal development, but unlike most other IAP-family proteins, survivin is virtually undetectable in normal adult human tissues. See Ambrosini et al. Nat. Med. 1997; 3(8):917-21.
Livin has been detected in some adult tissues and in embryonic tissues. Elevated levels of livin expression have been reported in melanomas, colon cancer cells, bladder cancer cells, and lung cancer cells. Two splice variants of livin have been reported, both of which contain a single BIR domain. The full length alpha variant has 298 amino acid residues, whereas the beta variant has 280 amino acid residues.
IAP-family proteins also have been identified in a number of species in addition to humans, including mammals such as the mouse, amphibians such as Xenopus species (African clawed toads), insects such as Drosophila species, and baculoviruses.
The ubiquitous and highly selective nature of survivin expression in cancer cells makes it a potentially useful diagnostic marker for cancer. For example, Rohayem et al. Cancer Res. 2000; 60:1815-17, have reportedly identified auto-antibodies to survivin in human lung and colorectal cancer patients.
Survivin has also been identified as a target for cancer therapy. The inhibiting effect of survivin on caspase-3 and caspase-7 has been implicated in the resistance of cancer cells to various apoptosis stimulating chemotherapeutic treatments. An antisense oligonucleotide that targets survivin expression has been reported to down-regulate survivin expression in an adenocarcinoma cell line and sensitize the cancer cells to the chemotherapeutic agent etoposide. See Olie et al. Cancer Res. 2000; 60:2805-9; and Mesri et al. J. Clinical Res., 2001; 108:981-990.
Cytokines are proteins and polypeptides produced by cells that can affect the behavior of other cells, such as cell proliferation, cell differentiation, regulation of immune responses, hematopoiesis, and inflammatory responses. Cytokines have been classified into a number of families, including chemokines, hematopoietins, immunoglobulins, tumor necrosis factors, and a variety of unassigned molecules. See generally Oxford Dictionary of Biochemistry and Molecular Biology, Revised Edition, Oxford University Press, 2000; and C. A. Janeway, P. Travers, M. Walport and M. Schlomchik, Immunobiology, Fifth Edition, Garland Publishing, 2001 (hereinafter Janeway and Travers). A concise classification of cytokines is presented in Janeway and Travers, Appendix III, pages 677-679, the relevant disclosures of which are incorporated herein by reference.
Hematopoietins include, for example erythropoietin, interleukin-2 (IL-2, a 133 amino acid protein produced by T cells and involved in T cell proliferation), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-13, IL-15 (a 114 amino acid IL-2-like protein, which stimulates the growth of intestinal epithelium, T cells, and NK cells), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), oncostatin M (OSM), and leukemia inhibitory factor (LIF).
Interferons include, for example, IFN-α, IFN-β, and IFN-γ (a 143 amino acid homodimeric protein produced by T cells and NK cells, which is involved in macrophage activation, increased expression of MHC molecules and antigen processing components, IG class switching, and suppression of TH2).
Immunoglobulins include, for example, B7.1 (CD80), and B7.2 (CD86), both of which co-stimulate T cell responses.
The tumor necrosis factor (TNF) family includes, for example, TNF-α, TNF-β (lymphotoxin), lymphotoxin-β (LT-β), CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BB ligand, Trail, and OPG ligand.
Various cytokines that are not assigned to a particular family include, for example, tumor growth factor-β (TGF-β), IL-1α, IL-1β, IL-1 RA, IL-10, IL-12 (natural killer cell stimulatory factor; a heterodimer having a 197 amino acid chain and a 306 amino acid chain, which is involved in NK cell activation and induction of T cell differentiation to TH1-like cells), macrophage inhibitory factor (MIF), IL-16, IL-17 (a cytokine production-inducing factor, which induces cytokine production in epithelia, endothelia, and fibroblasts), and IL-18.
Chemokines are a family of cytokines that are relatively small chemoattractant proteins and polypeptides, which stimulate the migration and activation of various cells, such as leucocyte migration (e.g., phagocytes and lymphocytes). Chemokines play a role in inflammation and other immune responses. Chemokines have been classified into a number of families, including the C chemokines, CC chemokines, CXC chemokines, and CX3C chemokines. The names refer to the number and spacing of cysteine residues in the molecules; C chemokines having one cysteine, CC chemokines having two contiguous cysteines, CXC having two cysteines separated by a single amino acid residue, and CX3C chemokines having two cysteines separated by three amino acid residues. Chemokines interact with a number of chemokine receptors present on cell surfaces. See Janeway and Travers, Appendix IV, page 680, the relevant disclosures of which are incorporated herein by reference.
In addition, chemokines can have immunomodulating activity and have been implicated in immune responses to cancer. For example, murine 6Ckine/SLC, the mouse analog of the human secondary lymphoid tissue chemokine (SLC), now commonly referred to as CCL21, has been reported to induce an antitumor response in a C-26 colon carcinoma tumor cell line. See Vicari, et al. J. Immunol. 2000; 165(4):1992-2000. Human CCL21 and its murine counterpart, 6Ckine/SLC, are classified as CC chemokines, which interact with the CCR7 chemokine receptor. Murine 6Ckine/SLC (muCCL21) is also reported by Vicari et al. to be a ligand for the CXCR3 chemokine receptor. Human CCL21, murine muCCL21 and a variety of other chemokines are implicated in the regulation of various immune system cells such as dendritic cells, T-cells, and natural killer (NK) cells.
Mig and IP-10 are CXC chemokines that interact with the CXCR3 receptor, which is associated with activated T cells. Lymphotactin is a C chemokine, which intereacts with the XCR1 receptor associated with T cells and NK cells. Fractalkine is a CX3C chemokine, which interact with the CX3CR1 receptor that is associated with T cells, monocytes and neutrophils.
NK cells are large granular lymphocytes that recognize and destroy cells that have been infected with a virus. NK cells can be regulated by interaction of immunomodulating polypeptide ligands with receptors on the NK cell surface. For example, ligands for the NKG2D receptor that can regulate NK cell activity, include chemokines such as muCCL21, and stress-inducible polypeptide ligands such as MHC class I chain-related antigens and UL16 binding proteins. Murine H60 minor histocompatibility antigen peptide is reported to bind to the NKG2D receptor, as well. See, e.g., Robertson et al. Cell Immunol. 2000; 199(1):8-14; Choi et al. Immunity 2002, 17(5):593-603, and Farag et al., Blood, 2002; 100(6):1935-1947.
The present invention fulfills an ongoing need for vaccines that can stimulate a general immune response against cancer cells by providing a DNA vaccine encoding a cancer-associated IAP-family protein and an immunoactive gene product in a single vector.